Inhibition of novel human-HPV hybrid ecDNA enhancers reduces oncogene expression and tumor growth in oropharyngeal cancer

Extrachromosomal circular DNA (ecDNA) have been found in most types of human cancers, and ecDNA incorporating viral genomes has recently been described, specifically in human papillomavirus (HPV)-mediated oropharyngeal cancer (OPC). However, the molecular mechanisms of human-viral hybrid ecDNA (hybrid ecDNA) for carcinogenesis remains elusive. We characterized the epigenetic status of hybrid ecDNA using HPVOPC cell lines and patient-derived tumor xenografts, identifying HPV oncogenes E6/E7 in hybrid ecDNA were flanked by novel somatic DNA enhancers and HPV L1 enhancers, with strong cis-interaction. Targeting of these enhancers by clustered regularly interspaced short palindromic repeats interference or hybrid ecDNA by bromodomain and extra-terminal inhibitor reduced E6/E7 expression, and significantly inhibited in vitro and/or in vivo growth only in ecDNA(+) models. HPV DNA in hybrid ecDNA structures are associated with novel somatic and HPV enhancers in hybrid ecDNA that drive HPV ongogene expression and carcinogenesis, and can be targeted with ecDNA disrupting therapeutics.


Introduction
Extrachromosomal circular DNA (ecDNA) represents a frequent driver of focal oncogene amplification during carcinogenesis, and the subsequent high expression of the genes carried on ecDNA [1][2][3] .This phenomenon is facilitated by the circular structure and open-chromatin configuration of ecDNA 4 , allowing transcriptional factors to easily access ecDNA.The unique spatial reorganization of ecDNA is thought to create new topological domains, providing opportunities for novel interactions between oncogenes and distant regulatory elements, and ecDNA mediated cis-interactions between enhancers and oncogenes ("enhancer hijacking,").Oncogenes residing on chromosomal DNA in the human genome may also be activated using so-called "mobile enhancers" from freefloating ecDNA 5 .Furthermore, ecDNA segments in close proximity may also interact through "ecDNA hubs" 6,7 .
Recent studies have shown that in Human papillomavirus (HPV) mediated cancers, human DNA and viral genomes can form circular human-viral hybrid ecDNA (hybrid ecDNA).HPV is an approximately 8kbp circular double-stranded DNA virus and includes oncogenes called E6 and E7 8,9 .HPV-mediated oropharyngeal cancer (HPVOPC) incidence has dramatically increased over the last 2 decades and has become one of the fastest growing cause of solid organ cancer death in the US 10,11 .In our previous analysis, hybrid ecDNA was identified in 16 out of 56 cases (around 30%) of HPVOPC 12 .While somatic integration of HPV oncogenes into the human genome is known as a carcinogenic driver of HPVOPC 13,14 , the molecular mechanisms by which hybrid ecDNA drives carcinogenesis have not been well described.Understanding these mechanisms and potential therapeutic vulnerabilities in hybrid ecDNA(+) HPVOPC are an important, unmet challenge in understanding and treating HPVOPC.
We hypothesized that in addition to oncogene amplification, HPV oncogene transcriptional upregulation via cis-interactions in hybrid ecDNA between enhancers and HPV oncogenes, and proximity of hybrid ecDNA and HPV episomal segments themselves are deeply involved in mediating the carcinogenic molecular functions of HPVOPC.
Here, we investigated the chromatin status of hybrid ecDNA using HPVOPC cell lines and PDX tumors and identified active enhancers that were newly created in hybrid ecDNA.The HPV oncogenes E6/E7 inside hybrid ecDNA were flanked by novel somatic enhancers in addition to novel HPV enhancers within the L1 region.HiC-seq identified a strong interaction between these enhancers and E6/E7, supporting a cis-interaction inside hybrid ecDNA.Inactivation of this enhancer by clustered regularly interspaced short palindromic repeats (CRISPR) interference reduced the expression of E6/E7 and proliferation.Furthermore, bromodomain and extra-terminal (BET) inhibitor treatment targeting hybrid ecDNA structures in ecDNA(+) tumors resulted in the significant inhibition of tumor growth in both cell lines and PDX tumors not seen in hybrid ecDNA(−) tumors, suggesting new avenues for therapeutic intervention in HPVOPC.

Identification of hybrid ecDNA in HPVOPC cell lines and PDX tumors
To determine hybrid ecDNA status, we performed WGS and RNA-seq on 2 HPVOPC cell lines, HMS001 and SCC154, and 2 patient-derived xenograft (PDX) tumors from clinical tumor samples of HPVOPC patients, described here as PDX_A and PDX_C.As negative control, we used the cell-line NOKSI, (spontaneously immortalized human cell line derived from normal oral mucosa that is HPV negative and known to not carry ecDNA).Analysis of WGS and RNA-seq data showed the presence of hybrid DNA molecules and hybrid transcripts in HMS001 and PDX_A but not in PDX_C, SCC154 or NOKSI.Using Amplicon Architect (AA), hybrid ecDNA was detected in HMS001 and PDX_A, but not in SCC154, PDX_B or NOKSI (Fig. 1A-D).Across samples, each sequence of hybrid ecDNA was unique, consistent with our previous analyses in clinical samples of HPVOPC 12 .Long read DNA-seq also detected structural variants for both viral and human segments on the same sequenced molecule, consistent with the AA results (Supplementary Fig. 1).For the cytogenetic validation of each hybrid ecDNA, multi-DNA FISH targeting each human genome and HPV genome on hybrid ecDNA was performed using a metaphase-spread in cell line and short-time cultured PDX tumors.
Overlapping of somatic and HPV probes was located outside of condensed chromosomes in HMS001 (hybrid ecDNA(+)) and PDX_A, confirming the existence of hybrid ecDNA (Fig. 1E and F), but no such overlapping probes were found in SCC154 (hybrid ecDNA-; Fig. 1G and H).We also observed HPV-only signals only in HMS001, PDX_A, and SCC154 showing the existence of episomal HPV DNA in each sample (Fig. 1E-G).The mean copy numbers of HPV/cell in HMS001, PDX_A, SCC154, and NOKSI were 4.14, 4.11, 2.3, and 0 respectively (Supplementary Table 1).

Novel enhancers are created in hybrid ecDNA structures
To elucidate chromatin status of hybrid ecDNA, we performed ChIP-seq for H3K27ac (activation mark), H3K4me1 (enhancer mark), and H3K4me3 (promoter mark), and ATAC-seq on HPVOPC cell lines and PDX tumors.Contrary to our expectations, previously described somatic enhancer regions were not hijacked, but clusters of H3K4me1 and H3K27ac peaks, indicative of active enhancers, were found in the regions with hybrid ecDNA (Fig. 2A).Intriguingly, a novel cluster of H3K4me1 and H3K27ac peaks was observed in HMS001, but not in SCC154 or NOKSI, suggesting the creation of a novel enhancer element in the hybrid ecDNA (Fig. 2A).Strikingly, the HPV integration site within hybrid ecDNA exists in the center of these novel enhancers in hybrid ecDNA.Specifically, H3K4me1 and H3K27ac enrichment was noted in the HPV L1 region within the hybrid ecDNA enhancer as part of a larger enhancer region and ATAC-seq confirmed these enhancers exist in open chromatin regions (Fig. 2B).In addition, the E6/E7 promoter was surrounded by these enhancers and making enhancepromoter complex.The same pattern was observed within the hybrid ecDNA region in PDX_A (Fig. 2C and D).Although the somatic genomic regions associated with each hybrid ecDNA were quite different and unique between HMS001 and PDX_A, we found that in each case, the E6/E7 promoter was surrounded by newly created enhancers in somatic regions and an enhancer in the HPV L1 region.Based on these data, we hypothesized that HPV gene expression, specifically E6/E7, was upregulated by newly created somatic human and HPV hybrid enhancer complexes on hybrid ecDNA .These phenomena only occurred in hybrid ecDNA samples, suggesting a hybrid ecDNAspecific mechanism.

Human and viral genomes on hybrid ecDNA interact directly with each other
To elucidate enhancer and HPV DNA interactions in hybrid ecDNA, HiC-seq was performed using the same cell lines and PDX tumors.Human somatic genomic sequences on hybrid ecDNA were divided into 2 segments (S1 and S2).The S1 enhancer region closely interacted with the HPV L1 region and the S2 enhancer region closely interacted with HPV E6/E7 regions in HMS001 (Fig. 3A and B).On the other hand, there was no such interaction in SCC154 that lacked hybrid ecDNA (Fig. 3C).This phenomenon was confirmed in PDX tumors as well (Fig. 3D-F).In PDX_A, the enhancer existed only in the S1 segment, and the S1 enhancer region closely interacted with HPV L1 and E6/E7 regions (Fig. 3D and E), and there was no such interaction in hybrid ecDNA-PDX_C (Fig. 3F).Although each hybrid ecDNA structure was unique, each of the novel somatic enhancer regions closely interacted with HPV genome, confirming the direct interaction of the human and viral genomes in hybrid ecDNA.

CRISPR interference targeting of enhancers on hybrid ecDNA blocks HPV oncogene expression
To elucidate the functional role of newly created enhancers on hybrid ecDNA, we performed CRISPR interference, targeting specific hybrid ecDNA enhancers in HMS001.
We generated dCas9-KRAB stable cell lines; HMS001, SCC154, and NOKSI.gRNAs targeting S1: the long part (gRNA#1) and the S2: the short part (gRNA#2) of the enhancer on hybrid ecDNA of HMS001 and nontarget controls were used (Fig. 4A).We confirmed the expression of dCas9 after doxycycline induction (Fig. 4B and C, Supplementary Fig. 2A and B).Consistent with our hypothesis, E6 and E7 expression were specifically reduced by the repression of the S2 enhancer region on hybrid ecDNA by CRISPR interference (CRISPRi) (Fig. 4D).This phenomenon was not seen in SCC154 or NOKSI without hybrid ecDNA (Supplementary Fig. 2C and D), supporting the notion that the enhancer on hybrid ecDNA was newly and uniquely created.Furthermore, compared to nontarget controls, S2 repression significantly inhibited the proliferation only in HMS001 (P=0.006) (Fig. 4E-G).This indicates that novel hybrid enhancer regions in hybrid ecDNA specifically drive expression of HPV oncogenes.

Hybrid ecDNA and HPV episomes are physically associated and can be targeted therapeutically in vitro and in vivo
Next, we investigated the potential for novel HPVOPC therapeutic strategies targeting hybrid ecDNA.We focused using the Bromo-and Extra-Terminal domain (BET) inhibitors as potential therapeutic agents that specifically target BRD4 as a key linker of ecDNA 6 .We hypothesized that disruption of the direct interaction of the enhancer in the somatic genome that interacts with HPV E6/E7 on hybrid ecDNA would inhibit HPV oncogene expression and reduce downstream gene targeting and proliferation.
To investigate the spatial properties of hybrid ecDNA in the cells, we performed multi-probe FISH using an EYA2 probe and an HPV probe in super resolution with and without JQ1 treatment for HMS001 cells.FISH signals for hybrid ecDNA and episomal HPV only tended to accumulate in the nucleus, and these signals were significantly reduced after JQ1 treatment.This suggests that the copy number of hybrid ecDNA and episomal HPV decreased after JQ1 treatment (Fig. 5A and B).
To investigate gene expression changes after JQ1 treatment, we at first checked the E6/E7 expression after JQ1 treatment (Fig. 6A).E6/7 expression was only downregulated in the hybrid ecDNA(+) HMS001 cell line-not in hybrid ecDNA(−) SCC154.MYC is one of the key oncogenes regulated by BRD4, and as expected, MYC was downregulated by JQ1 even in hybrid ecDNA(−) cell lines.(Fig. 6B, C, and Supplementary Fig. 3).In addition, upon JQ1 treatment, E6/E7 expression was reduced in a concentrationdependent manner (100nM and 1μM) at 6h after JQ1 treatment and more rapidly at 24 h (Fig. 6B and C).A similar result was seen in proliferation assays in HMS001 and SCC154, in which JQ1 treatment significantly inhibited tumor growth only in hybrid ecDNA(+) HPVOPC, but not in ecDNA(−) HPVOPC (*P = 0.03, P = 0.12, respectively) Nakagawa T, et al.
(Fig. 6D).Comparison of ChIP-seq data between JQ1 treatment and DMSO in HMS001 indicated that while we found many BRD4 peaks significantly reduced after JQ1 treatment, we found many H3K27ac peaks significantly enriched after JQ1 treatment (Supplementary Fig. 4A and 4B).To clarify specific pathways affected by chromatin alterations, 810 differentially upregulated H3K27ac peaks along with H3K4me1 peaks (FC>2 and Ifdr<0.3)were identified.Gene Ontology analysis showed that GO terms reported to be suppressed by HPV infection were restored, such as, "epithelial cell differentiation" and "Apoptosis" (Supplementary Fig. 4C).On the other hand, HiC-seq showed no significant changes after JQ1 treatment, suggesting that the structure of the hybrid ecDNA itself is not affected by JQ1 treatment (Supplementary Fig. 4D and 4E).
We investigated JQ1 treatment in an in vivo model using a patient-derived xenograft model of HPVOPC (Fig. 7A) with hybrid ecDNA(+) (PDX_A) and hybrid ecDNA(-) (PDX_C and PDX004).In the PDX_A JQ1 treatment group, tumor growth was significantly inhibited compared to the vehicle control group (tumor volume: P = 2x10 -5 , tumor weight: P = 1x10 -4 respectively) (Fig. 7B-F).Consistent with our hypothesis, E6/E7 expression was also reduced after JQ1 treatment in this model (P < 1x10 -4 ) (Fig. 7G).On the other hand, in PDX_C and PDX004: hybrid ecDNA-models, JQ1 did not significantly inhibit tumor growth (Fig. 7H-M), suggesting specific targeting of hybrid ecDNA(+) tumors.Furthermore, multi-FISH targeting hybrid ecDNA using a short-time culture cell line from PDX_A tumors showed reduction of hybrid ecDNA FISH signals after JQ1 treatment (Supplementary Fig. 5A-C) and ChIP-seq results also identified changes in 496 H3K27ac peaks along with H3K4me1 peaks (FC>2 and Ifdr<0.3).GO analysis of genes associated with these changes include "epithelial cell differentiation", and "regulation of p38MAPK cascade" (Supplementary Fig. 6A-C).HiC-seq showed no significant changes after JQ1 treatment in PDX A as well as in HMS001 (Supplementary Fig. 6D and 6E).A total of 248 K27ac peaks along with K4me1 peaks were common to hybrid ecDNA(+) cell line and PDX treated with JQ1, again including GO terms such as "epithelial cell differentiation" and "positive regulation of apoptotic process", suggesting changes associated with HPV inhibition (Supplementary Fig. 7).Taken together, these data suggest the potential for specific therapy targeting hybrid ecDNA through interruption of HPV mediated gene expression changes.

Discussion
While the formation of ecDNA is commonly associated with DNA damage, such as chromosome shattering (chromothripsis), and breakage-fusion-bridge cycles (BFBs) 2,3,15,16 , viral integration into the human genome also induces genomic instability 17,18,19,20 and causes formation of hybrid ecDNA and carcinogenesis.Although HPV integration often occurs in non-coding regions and some hot spots as previously reported, the location of integration is thought to be random 21,22 .Furthermore, HPV integration is reported to change chromatin accessibility status and activate surrounding genes 23,24 , but the mechanisms behind this phenomenon are yet to be elucidated.Although HPV integration around originally existing enhancers, and hijacking of known enhancers has been reported previously 25 , we have observed that HPV integration creates novel enhancers in ecDNA that did not exist in OPC samples without HPV integration at that locus.We found HPV genome integration sites were surrounded by newly created enhancers and these regions formed hybrid ecDNA in both HPVOPC cell lines and PDX tumors from clinical HPVOPC patients.In addition, we also demonstrated the HPV genome itself can serve as an enhancer region within ecDNA.This is perhaps one reason why HPV integration may be seen as "random" or not biased to integration with known enhancer/promoter regions: HPV genome sequences themselves can serve as enhancers and can induce enhancer activity in genome regions that do not serve as enhancers in the normal physiologic state.
In our study, cis-interaction between HPV and newly created enhancers on hybrid ecDNA was confirmed by HiC-seq, and CRISPRi targeting this enhancer reduced the expression of E6/E7 and proliferation consistently.Interestingly, in HMS001, HPV is surrounded on both sides by newly created enhancers, but only one enhancer (S2) showed inhibition of HPV E6/E7 expression and cell growth.This is consistent with the interaction in HiC-seq data, which also showed strong interaction between S2 and E6/E7 in HMS001.In this case, the S1 enhancer was working the enhancer complex with L1 enhancer of HPV.We also made the novel observation that HPV genome regions, including the L1 region, can serve as enhancers when integrated into hybrid ecDNA structures.Detailed examination of the hybrid ecDNA structure along with the ChIP-seq data shows that an enhancer of HPV in cell line (HMS001) or PDX models, forms a complex with an enhancer in the L1 region of HPV.This is consistent with the fact that the suppression of HPV E6/E7 expression did not occur by repression of the enhancer on the human side alone.Although the cell lines and PDX from different patients had completely different hybrid ecDNAs, they shared the composition of newly created enhancers as well as HPV L1 enhancers flanking E6/E7, suggesting that L1 regions might be key enhancer regions driving HPV expression in hybrid structures.Furthermore, we explored the role of hybrid ecDNA association with episomal HPV in HPVOPC.Although it was difficult to specifically define 'hybrid ecDNA hubs' in our analysis, hybrid ecDNAs and episomal HPV were found nearby each other in the nucleus, and HPV E6/E7 expression was strikingly reduced after JQ1 treatment.It is reasonable that the copy number of HPV in hybrid ecDNA is not as high as copy number of the oncogene in other cancer types 6 , because hybrid ecDNA can drive high levels of HPV transcription by coexisting with episomal HPV.
In the past few years, development of therapies that target ecDNA has been accelerating 26 .In this study, we identified that targeting the enhancer on hybrid ecDNA by CRISPRi reduced the expression of E6/E7 and proliferation in vitro model in HPVOPC.Furthermore, we showed that specific targeting of hybrid ecDNA with a BET inhibitor in HPVOPC was able to effectively reduce tumor growth and oncogene expression in both in vivo and in vitro models.We have now seen in this study using the first preclinical model targeted at hybrid ecDNA, that BET inhibitors are a potential new targeted therapy for patients with hybrid ecDNA(+) tumors.This provides a rationale for biomarker driven targeting of hybrid ecDNA(+) HPVOPC, and investigation of other types of treatment for ecDNA(+) HPVOPC that specifically targets ecDNA.

Clinical samples
Clinical specimens for whole-genome sequencing (WGS) and RNA-seq were collected from HPVOPC patients who underwent surgery at Jacobs Medical Center at the University of California, San Diego (UCSD) Health.Written informed consent was obtained from all patients.These samples were shared with the UCSD Human Research Protection Program (institutional review board (IRB)-approved protocol HRPP# 181755) by way of the Moores Cancer Center Biorepository and Tissue Technology resource.Two independent pathologists confirmed that the purity of the primary tumor was at least 80%.
HPV status was determined by p16 immunohistochemistry or in-situ hybridization.

Patient-derived xenografts (PDX)
Female 4-week-old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from The Jackson Laboratory.Fresh clinical HPVOPC tumor samples were extracted at surgery and cut into approximately 3mm pieces and transplanted into 6-8-week-old female NOD/SCID mice.Xenografting procedures were described previously 27,28 .

Cell culture
SCC154 was purchased from the American Type Culture Collection (ATCC).
For short-time cell culture of PDX tumors, PDX mice were euthanized for tissue retrieval, and tumors were dissected.Then, tumors were digested and isolated for shorttime cell culture.Procedures were described elsewhere 29 .Cells were grown in defined keratinocyte serum free media (Invitrogen, Carlsbad, CA) supplemented with 1% antibiotics, 5 ng/ml mouse epidermal growth factor (Invitrogen) and 2 × 10 −11 M cholera (Sigma-Aldrich) at 37 °C with 5% CO2.

Whole genome sequencing
DNA was extracted using the QIAquick DNA mini kit (Qiagen, Hilden, Germany) for high-quality extraction per the manufacturer instructions.Library preparation and sequencing was performed with the Illumina NovaSeq 6000 at the UCSD IGM Genomics Center.Hg38 and HPV genome sequences (accession number: AY686584.1)were used for reference genome.
In brief, CNVkit v0.9.9 was employed for copy number segmentation and estimation.
Segments with copy number ≥ 2.5 copies above chromosome arm ploidy, as well as viral genome regions with CN>=1 were extracted using the AmpliconSuite-pipeline and used as seed regions for analysis of focal amplifications.For each seed region, AmpliconArchitect searched the region and nearby loci for discordant read pairs, which are indicative of genomic structural rearrangements.Genomic segments are defined based on the positions of gene breakpoints and changes in copy number.AmpliconArchitect utilizes structural variant signatures, such as discordant paired-end reads and CNV Nakagawa T, et al.
boundaries, to partition all intervals into segments.It then constructs an amplicon graph based on these segments and decomposes the graph into genome paths and cycles that explain the observed changes in copy number and structural variation.The AmpliconClassifier script was used to classify amplicons into categories such as ecDNA, breakage-fusion-bridge, complex non-cyclic, linear, and no focal amplification based on rules related to patterns of structural variation, copy number and decomposed genome paths from AmpliconArchitect.Circular visualizations of ecDNA genome structure and annotation tracks were generated using CycleViz (https://github.com/AmpliconSuite/CycleViz) 31.

RNA extraction and RNA sequencing
RNA was extracted following the protocol of the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany).RNA concentration, purity, and integrity were verified

ATAC-seq
The precise protocol was previously shown 32  Tagmentation enzyme (Illumina) at 37 °C for 60 min with shaking 500 rpm.The tagmentated DNA was purified using MinElute PCR purification kit (Qiagen).The libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB, Ipswich, MA) with primer extension at 72°C for 5 min, denaturation at 98°C for 30s, followed by 8 cycles of denaturation at 98°C for 10 s, annealing at 63°C for 30 s and extension at 72°C for 60 s.Amplified libraries were then purified using MinElute PCR purification kit (Qiagen), and two size selection steps were performed using SPRIselect bead (Beckman Coulter, Brea, CA) at 0.55X and 1.5X bead-to-sample volume rations, respectively.
Final libraries were quantified using Qubit (Thermo Fisher Scientific) and checked for library size distribution using 4200 TapeStation (Agilent Technologies, Santa Clara, CA).
Library preparation and sequencing was performed with the Illumina NovaSeq 6000 at the UCSD IGM Genomics Center.

ChIP-seq and ATAC-seq analysis
Raw sequence data were first trimmed for sequencing adapters using fastp 33 , then aligned to human genome (hg38) augmented by the HPV type 16 genome sequence as an extra chromosome (accession number AY686584.1),using STAR aligner 34 with chimeric alignments allowed in the output.Peak calling was done in an unbiased way on an artificial sample created as the union of reads sampled randomly (with equal weight per sample) from all samples of the same type (ATAC-seq, H3K27ac, etc.), using HOMER (Hypergeometric Optimization of Motif EnRichment) 35 .Peak quantitation was done for each sample individually by counting reads aligning to the peak regions determined in the previous step.

Peak normalization and differential analysis
Raw read numbers in peak regions of all samples of the same type were normalized separately.For the H3K27ac mark, we have two replicates each of HMS001 and PDX_A treated with either vehicle or JQ1 (8 samples total).Normalization proceeds under the assumption that the tallest peaks should be comparable across samples.To this end, we found peak regions in which the raw peak values are among the top 30-percentile for each sample.To this subset of all peak regions, we apply the Relative Log Expression (RLE) normalization method 36 .The normalization factors per sample are then applied to all peak regions.This normalization method is insensitive to potentially different levels of low-level "background" signal in different samples.Differentially acetylated regions (H3K27ac mark) were identified as follows: Since we have only two biological replicates per condition, and ~10 4 tests to carry out, the standard t-test has very low power.Instead, for each peak region and each comparison we calculate a z-score, in which the standard deviation in the denominator is modeled as a function of mean signal across samples being compared.This function is obtained as the loess regression fit through the calculated standard deviation data versus mean signal, using all eight samples together as if they were replicates.The approximate z-scores in every comparison are then assessed for significance by the empirical Bayes method 37 .The result is a posterior error probability lfdr assigned to each peak region in every comparison.

Finding significantly differentially activated peaks (H3K27ac) within active promoters (H3K4me3) and enhancers (H3K4me1)
We look for intercept of significantly differentially activated genomic regions (H3K27ac peaks with significance operationally defined as lfdr < 0.3) with a) peak regions defined by the active promoter mark H3K4me3 or b) by the enhancer mark H3K4me1.

HiC library preparation and sequencing
HiC experiments were performed based on the protocol of Arima-HiC Kit (Arima Genomics, Carlsbad, CA).Briefly, chromatin obtained from each cell line or PDX tumor was first cross-linked and digested using a restriction enzyme cocktail.The digested ends were labeled with biotinylated nucleotides and ligated to capture the sequence and structure of the genome.The ligated DNA was purified and fragmented, and the enriched biotinylated fragments were subjected to a custom library preparation protocol using an Arima Library preparation module.Sequencing was performed with the Illumina NovaSeq 6000 by Arima Genomics.

Hi-C data processing.
Hi-C data were processed by the runHiC python package (https://pypi.org/project/runHiC/)with a custom reference genome composed of human genome assembly hg38 augmented by the HPV type 16 genome as an extra chromosome.
The runHiC package can remove duplicate reads, assign reads to restriction fragments, filter out invalid interaction pairs, and generate binned interaction matrices in *.mcool format.We specified the enzyme name as Arima in the filtering step to remove the read pair that maps to the same restriction fragment.We used a bin size as small as 1kbp in the binning step to generate a high-resolution interaction matrix for each chromosome.
Given the primary ecDNA structure predicted by AmpliconArchitect (and validated by long reads), we extracted the submatrices from each genomic interval composing the ecDNA and assembled these submatrices into one matrix corresponding to ecDNA according to the order and orientation of the corresponding intervals on the ecDNA.
Finally, we applied ICE normalization on the resulting matrix and visualized them in Fig.

Metaphase chromosome spreads
Cultured cells were enriched in metaphase by treatment with KaryoMAX Colcemid (Gibco) at 100 ng ml −1 overnight.After washing once with PBS, single-cell suspensions were incubated in 75 mM KCl for 15 min at 37°C.Carnoy's fixative (3:1 methanol:glacial acetic acid) was used for cell fixation and cells were spun down.Cells were washed 3 more times with fixative solution and dropped onto humidified glass slides.

Fluorescence in situ hybridization (FISH)
Fixed cells on slides from cell lines or primary cultured cells from PDX were equilibrated in 2xSCC buffer and dehydrated in 70%, 85% and 100% ethyl alcohol for approximately 2 min each.FISH probes for human genome parts of hybrid ecDNA (EYA2 and NDUFC1 FISH probe) were purchased from EMPIRE genomics (Depew, NY), and HPV16 probes were purchased from Arbor Biosciences (Ann Arbor, MI).
Diluted FISH probes in hybridization buffer were added to the sample and covered with a coverslip.Slides were denatured at 72 °C for 1 min and hybridized overnight at 37 °C.
The slides were then washed with 0.4× SSC, and 2× SSC-0.1% Tween 20.DAPI was applied to the samples for 1 min.before washing again and mounting with Prolong Gold.

Microscopy
Confocal images were collected on a Nikon confocal plus STORM system using a 100 x 1.49 NA TIRF objective (Moores Cancer Center Shared Resources).For STORM, samples were mounted in STORM buffer (50 mM Tris, pH 8.0, 10 mM NaCl, 10% glucose, 0.1 M mercaptoethanolamine 56 units/ml glucose oxidase, and 340 units/ml catalase) and duplicate images were collected at the same pixel size (0.16 micrometers and 256x256 field of view) in the confocal mode and in the super-resolution mode using an ANDOR IXON3 Ultra DU897 EMCCD camera to enable overlay of the confocal (DAPI) and STORM images.For super-resolution images, TIRF illumination settings were used as appropriate to enhance the signal-to-noise ratio.The images were collected at frame rate (about 15 millisecond exposure time using a 256x256 pixel area of the camera chip) using the sequential illumination setting in the STORM acquisition module in Nikon Elements software (version 4.6).Laser power was adjusted so that 50-350 localization events were recorded per channel in each 256x256 pixel area frame.Acquisition was stopped once 1 -3 million localization events were recorded.
Analysis of the image stacks was carried out using the STORM analysis module of the Elements software.The STORM images were superimposed on the confocal DAPI images for context.

BET inhibitor treatment for cell lines
The effect of BET inhibitor was investigated on cell line HMS001 and SCC154.
Proliferation was investigated in the presence of DMSO (negative control), as well as JQ1 (10nM, 100nM, and 1μM) (SML1524, Sigma-Aldrich).Cells were seeded in 96-well plates at a density of 8,000 cells/well for HMS001 and 4,000 cells/well for SCC154.
Relative absorbance was measured at day 0 and 2 days after the JQ1 or DMSO treatment.

BET inhibitor treatment for PDX
When the tumor volume of PDX reached about 150-250 mm 3 , these mice were randomly divided into 2 groups (each group, n=6).Mice were treated daily with either JQ1 at 50mg/kg IP or vehicle control.Tumor samples were harvested after 2 weeks of JQ1 treatment.To confirm each gene expression after treatment, quantitative PCR for E6/E7 and ACTB was performed (Supplemental Table 2) Nakagawa T, et al.

Statistical analysis
Comparison of the results of qPCR, proliferation assay, PDX tumor volume, and PDX tumor weight in each group was analyzed by Student's t-test using GraphPad Prism version 10.1.

Data access
WGS, long read DNA-seq, RNA-seq, ChIP-seq, ATAC-seq, HiC-seq of each cell line and PDX tumor data were submitted to the GEO database under the accession number ○○ (We are in the process of registration, and will update once we got the numbers).
Nakagawa T, et al.Cis-interactions between enhancer and HPV in hybrid ecDNA were analyzed by HiC-seq.Human genome regions on hybrid ecDNA were divided into 2 segments (S1 and S2).The S1 enhancer region closely interacted with the HPV L1 region, and the S2 enhancer region closely interacted with the HPV E6/E7 regions in HMS001 (black arrow in A and B).On the other hand, there was no such interaction in SCC154 that lacked hybrid ecDNA (C).This phenomenon was confirmed in PDX tumors (D-F).In PDX_A, the enhancer existed only in the S1 segment, and the S1 enhancer region closely interacted with the HPV L1 and E6/E7 regions in PDX_A (black and yellow arrows in D and E).On the other hand, there was no such interaction in PDX_C that lacked hybrid ecDNA (F).

Figure legends
Although each hybrid ecDNA structure was unique, each of the novel human enhancer regions closely interacted with HPV, confirming the direct interaction of the human and viral genomes on the hybrid ecDNAs.Fig. 4  Multi-probe FISH, using an EYA2 probe and an HPV probe on hybrid ecDNA on super resolution DMSO and JQ1 treatment of HMS001 cells, is shown (A and B).Hybrid ecDNA was observed nearby in the nucleus along with episomal HPV in the "no treatment" condition (A).Each signal (green: EYA2, red: HPV, and blue: DAPI) were also shown separately (bottom).(A).FISH signals of hybrid ecDNA and episomal HPV were reduced after JQ1 treatment (B).Scale bar shows 5m (0.2m in expanded picture).Cartoon illustrating how hybrid ecDNA hub disruption may decrease transcription is illustrated (right in A and B). , respectively) at 24h (B).MYC and E6/E7 expressions were reduced in a concentration-dependent manner in western blotting at 6h and 24 h after JQ1 treatment (C).Proliferation assay using JQ1 for HMS001 and SCC154 were shown.JQ1 treatment significantly inhibited tumor growth only in HMS001 in 1uM, but not in SCC154 (*P = 0.03, P = 0.12, respectively) (D).Fig. 7: JQ1 inhibited proliferation only in hybrid ecDNA(+) HPVOPC PDX tumors.JQ1 treatment was performed in HPVOPC PDX models.Six PDX_A (hybrid ecDNA+) mice were divided into a vehicle control group and a JQ1 treatment group.Each mouse possessed tumors in both flanks (A).Tumors were harvested after 2 weeks of vehicle control or JQ1 treatment (B).Tumors were harvested after 2 weeks of JQ1 treatment or vehicle control.Tumor volumes of each condition are shown (C-E).In the PDX_A JQ1 treatment group, tumor growth was significantly inhibited compared to the vehicle control group (tumor volume: P = 2x10 -5 , tumor weight: P = 1x10 -4 respectively) (C and F).qPCR of E6/E7 is shown between JQ1 treatment and vehicle control.E6/E7 expression was also reduced after JQ1 treatment (P < 1x10 -4 ) (G).JQ1 treatment for hybrid ecDNA-HPVOPC PDX (PDX_C and PDX004) was also performed (H-M).Neither tumor volume nor tumor weight were inhibited significantly compared to the vehicle control group in PDX_C (H-J) and PDX004 (K-M).
using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA).In addition, an RNA Integrity Number (RIN) of 7.0 or greater by TapeStation was required for quality assessment.Library preparation and RNA sequencing were performed by the UCSD IGM Genomics Center utilizing an Illumina NovaSeq 6000.Long read DNA-sequencing High molecular weight DNA(>40kb) was extracted using the Nanobind CBB kit or Nanobing tissue kit (PacBio, Menlo Park, CA) following manufacturer's recommendations.Long read DNA sequencing was performed using 1 SMRT Cell per sample.Library preparation and sequencing were performed by the UCSD IGM Genomics Center with the Illumina NovaSeq 6000.

Fig. 1 :Fig. 2 :Fig. 3 :
Figure legendsFig.1:Identification of hybrid ecDNA in HPVOPC using AA and FastViFI and validation of hybrid ecDNA by multi-FISH.Examples of circular hybrid ecDNA suggested by AA results in HMS001 (A and C) and PDX tumor PDX_A (B and D).Multi-FISH using each ecDNA specific probe (green) and HPV specific probe (red) for metaphase spread cells showed overlapping of each probe signal in the same place only in hybrid ecDNA(+) samples (yellow allow in E and F).Hybrid ecDNA(+) samples also showed the red HPV signals alone (white arrowhead in E and F), suggesting HPV-only episomes.Hybrid ecDNA(-) cell line SCC154 only showed the red HPV signal (white arrowhead in G).Control cell line NOKSI did not show any signal (H).Scale bar shows 10m.Fig.2: Detecting active enhancer using ChIP-seq, and new identification of HPV integration mechanism in hybrid ecDNA.ChIP-seq results of Input, K4me3 (promoter), K4me1 (enhancer), and K27ac (activation mark) for NOKSI (normal control), SCC154 (hybrid ecDNA-HPVOPC) and HMS001 (hybrid ecDNA+ HPVOPC) were shown.Promoter is indicated by the light green box, enhancers by the yellow box, and the sequences included in hybrid ecDNA of HMS001 by the light blue box.The active enhancer is marked by the red oval (top) (A).Expanded hybrid ecDNA sequence tracks are shown (bottom), and include anexpanded enhancer map showing HPV integration occurred in the exact center of the active enhancer mark (bottom) (A).The HPV integration site and its ChIP-seq results are also shown.On the other hand, active enhancers already existing originally were not included in hybrid ecDNA (bottom) (A).(B)Hybrid ecDNA in HMS001with ATAC-seq (top) and ChIPseq(bottom) are shown in the CycleViz plot.A related figure using NOKSI (normal control), PDX_C (hybrid ecDNA-HPVOPC), and PDX_A (hybrid ecDNA+ HPVOPC) is shown (C and D).The newly created active enhancer made a complex with 2 promoters (bottom) (C).Fig.3: Human and viral genomes on hybrid ecDNA interacted directly with each other.Cis-interactions between enhancer and HPV in hybrid ecDNA were analyzed by HiC-seq.Human genome regions on hybrid ecDNA were divided into 2 segments (S1 and S2).The S1 enhancer region closely interacted with the HPV L1 region, and the S2 enhancer region closely interacted with the HPV E6/E7 regions in HMS001 (black arrow in A and B).On the other hand, there was no such interaction in SCC154 that lacked hybrid ecDNA (C).This phenomenon was confirmed in PDX tumors (D-F).In PDX_A, the enhancer existed only in the S1 segment, and the S1 enhancer region closely interacted with the HPV L1 and E6/E7 regions in PDX_A (black and yellow arrows in D and E).On the